G protein coupled receptors (GPCRs) transmit extracellular signals into cells via intracellular G protein heterotrimers (1). Of currently marketed drugs, more than 30% modulate GPCRs (2). Only 10% of the 367 human endogenous ligand GPCRs are targeted by current drugs, leaving many future targets. Novel modes of defining the activity of ligands that bind to GPCRs could contribute to the treatment of human disease.
The response to epinephrine or adrenaline is a prototypic GPCR action. Equilibrium binding studies in frog erythrocyte membranes demonstrated homogeneous binding for antagonists, while agonists exhibited two states of agonist affinity (3). The ternary complex model of agonist, receptor, and G protein accounts for the ternary complex exhibiting a higher agonist affinity than the binary complex (4). Adenylyl cyclase assays define the intrinsic activity, or efficacy, for each compound. Receptors in the high affinity state range from 50% for agonists of the lowest intrinsic activity to 95% for full agonists, the percent correlated roughly with the intrinsic adenylyl cyclase activity of the agonist. The functional consequences of cellular ternary complex formation include the rapid binding of GTP to the Gα subunit, release of the receptor and the Gβγ dimer, and exposure of new Gα and Gβγ surfaces to interact with effectors such as adenylyl cyclase (5). Ternary complex formation for a series of agonists is expected to correlate with adenylyl cyclase activities. More detailed ternary complex formulations take into account the idea that receptors can exist in different activity states (6).
The present inventors have previously studied the formyl peptide receptor and its numerous fluorescent ligands. The solublilized receptor forms a high agonist affinity complex with G proteins and arresting. See, Bennett, et al., 2001 J. Biol. Chem. 276, 22453–22460; Bennett, et al., 2001, J. Biol. Chem. 276, 49195–49203; and Key, et al., 2001, J. Biol. Chem. 276, 49204–49212. Beads derivatized with chelated nickel bind hexahistidine-tagged G protein heterotrimers, and as shown by flow cytometry, form ternary complex with FPR constructs on G protein beads. The constructs included wild type receptor detected with fluorescent ligand, receptor-Gα fusion protein detected with fluorescent ligand, and receptor-GFP fusion protein detected with nonfluorescent ligand See, Simons, et al., 2003, Mol. Pharmacol. 64, 1227–1238. The technology demonstrated also includes one of the receptors for adrenaline, the beta 2 adrenergic receptor (β2AR), which was used in the form of a fusion protein with green fluorescent protein, the β2AR-GFP fusion protein. A well-known β2AR ligand was attached to beads, and the formation of ligand:receptor binary complexes was detected by increasing fluorescence of the beads, in a process analogous to affinity chromatography. As with the FPR-GFP, β2AR-GFP was able to bind cognate agonists to form agonist:receptor-GFP binary complexes, which in turn were able to bind to cognate hexahistidine-tagged G protein on chelated nickel beads to form ternary agonist:receptor-GFP:G protein complexes on chelated nickel beads. These complexes were also detected by increasing fluorescence of the beads.
The interactions of ligands with GPCRs have mainly been studied in membrane preparations with radiolabelled ligands (e.g., see references 3 and 4 for the β2AR), which are not amenable to high throughput screening. Therefore, appropriate tools in these areas have been limited and, in many situations, unsatisfactory. Thus, there is a need for a homogeneous, small volume bead-based approach compatible with high throughput flow cytometry, which would allow evaluation of G protein coupled receptor molecular assemblies. The present approach provides a general solution for addressing that need.